Article

Role of protein transamidation in serotonin-induced proliferation and migration of pulmonary artery smooth muscle cells.

Rutgers University, Piscataway, New Jersey, USA.
American Journal of Respiratory Cell and Molecular Biology (Impact Factor: 4.11). 04/2011; 44(4):548-55. DOI: 10.1165/rcmb.2010-0078OC
Source: PubMed

ABSTRACT Pulmonary hypertension is characterized by elevated pulmonary artery pressure and pulmonary artery smooth muscle cell (SMC) proliferation and migration. Clinical and experimental evidence suggests that serotonin (5-HT) is important in these responses. We previously demonstrated the participation of the 5-HT transporter and intracellular 5-HT (5-HTi) in the pulmonary vascular SMC-proliferative response to 5-HT. However, the mechanism underlying the intracellular actions of 5-HT is unknown. We speculated that 5-HTi activates SMC growth by post-translational transamidation of proteins via transglutaminase (TGase) activity, a process referred to as serotonylation. To test this hypothesis, serotonylation of pulmonary artery SMC proteins, and their role in 5-HT-induced proliferative and migratory responses, were assessed. 5-HT caused dose- and time-dependent increase in serotonylation of multiple proteins in both bovine and rat pulmonary artery SMCs. Inhibition of TGase with dansylcadaverin blocked this activity, as well as SMC-proliferative and migratory responses to 5-HT. Serotonylation of proteins also was blocked by 5-HT transporter inhibitors, and was enhanced by inhibition of monoamine oxidase, an enzyme known to degrade 5-HTi, indicating that 5-HTi levels regulate serotonylation. Immunoprecipitation assays and HPLC-mass spectral peptide sequencing revealed that a major protein serotonylated by TGase was fibronectin (FN). 5-HT-stimulated SMC serotonylation and proliferation were blocked by FN small interfering (si) RNA. These findings, together with previous observations that FN expression in the lung strongly correlates with the progression of pulmonary hypertension in both experimental animals and humans, suggest an important role of FN serotonylation in the pathogenesis of this disease.

0 Followers
 · 
66 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Tissue transglutaminase 2 (TG2) is a multifunctional enzyme that cross-links proteins with monoamines such as serotonin (5-hydroxytryptamine, 5-HT) via a transglutamidation reaction, and is associated with pathophysiologic vascular responses. 5-HT is a mitogen for pulmonary artery smooth muscle cells (PASMCs) that has been linked to pulmonary vascular remodeling underlying pulmonary hypertension development. We previously reported that 5-HT-induced PASMC proliferation is inhibited by the TG2 inhibitor monodansylcadaverine (MDC); however, the mechanisms are poorly understood. In the present study we hypothesized that TG2 contributes to 5-HT-induced signaling pathways of PASMCs. Pre-treatment of bovine distal PASMCs with varying concentrations of the inhibitor MDC led to differential inhibition of 5-HT-stimulated AKT and ROCK activation, while p-P38 was unaffected. Concentration response studies showed significant inhibition of AKT activation at 50μM MDC, along with inhibition of the AKT downstream targets mTOR, p-S6 kinase and p-S6. Furthermore, TG2 depletion by siRNA led to reduced 5-HT-induced AKT activation. Immunoprecipitation studies showed that 5-HT treatment led to increased levels of serotonylated AKT and increased TG2-AKT complex formations which were inhibited by MDC. Overexpression of TG2 point mutant cDNAs in PASMCs showed that the TG2 C277V transamidation mutant blunted 5-HT-induced AKT activation and 5-HT-induced PASMC mitogenesis. Finally, 5-HT-induced AKT activation was blunted in SERT genetic knock-out rat cells, but not in their wild-type counterpart. The SERT inhibitor imipramine similarly blocked AKT activation. These results indicate that TG2 contributes to 5-HT-induced distal PASMC proliferation via promotion of AKT signaling, likely via its serotonylation. Taken together, these results provide new insight into how TG2 may participate in vascular smooth muscle remodeling.
    Cellular Signalling 09/2014; 26(12). DOI:10.1016/j.cellsig.2014.09.002 · 4.47 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Serotonin (5-HT) induces concentration-dependent metabolic effects in diverse cell types, including neurons, entherochromaffin cells, adipocytes, pancreatic beta-cells, fibroblasts, smooth muscle cells, epithelial cells, and leukocytes. Three classes of genes regulating 5-HT function are constitutively expressed or induced in these cells: (a) membrane proteins that regulate the response to 5-HT, such as SERT, 5HTR-GPCR, and the 5HT3-ion channels; (b) downstream signaling transduction proteins; and (c) enzymes controlling 5-HT metabolism, such as IDO and MAO, which can generate biologically active catabolites, including melatonin, kynurenines, and kynurenamines. This review covers the clinical and experimental mechanisms involved in 5-HT-induced immunomodulation. These mechanisms are cell-specific and depend on the expression of serotonergic components in immune cells. Consequently, 5-HT can modulate several immunological events, such as chemotaxis, leukocyte activation, proliferation, cytokine secretion, anergy, and apoptosis. The effects of 5-HT on immune cells may be relevant in the clinical outcome of pathologies with an inflammatory component. Major depression, fibromyalgia, Alzheimer disease, psoriasis, arthritis, allergies, and asthma are all associated with changes in the serotonergic system associated with leukocytes. Thus, pharmacological regulation of the serotonergic system may modulate immune function and provide therapeutic alternatives for these diseases.
    Journal of Immunology Research 01/2015; 2015:1-21. DOI:10.1155/2015/354957 · 2.93 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Human coronary artery smooth muscle cells (HCASMCs) play an important role in the pathogenesis of coronary atherosclerosis and coronary artery diseases (CAD). Serotonin is a mediator known to produce vascular smooth muscle cell mitogenesis and contribute to coronary atherosclerosis. We hypothesize that the HCASMC possesses certain functional constituents of the serotonergic system such as: tryptophan hydroxylase and serotonin transporter. Our aim was to examine the presence of functional tryptophan hydroxylase-1 (TPH1) and serotonin transporter (SERT) in HCASMCs. The mRNA transcripts by qPCR and protein expression by Western blot of TPH1 and SERT were examined. The specificity and accuracy of the primers were verified using DNA gel electrophoresis and sequencing of qPCR products. The functionality of SERT was examined using a fluorescence dye-based serotonin transporter assay. The enzymatic activity of TPH was evaluated using UPLC. The HCASMCs expressed both mRNA transcripts and protein of SERT and TPH. The qPCR showed a single melt curve peak for both transcripts and in sequence analysis the amplicons were aligned with the respective genes. SERT and TPH enzymatic activity was present in the HCASMCs. Taken together, both TPH and SERT are functionally expressed in HCASMCs. These findings are novel and represent an initial step in examining the clinical relevance of the serotonergic system in HCASMCs and its role in the pathogenesis of coronary atherosclerosis and CAD.
    Molecular Biology Reports 04/2015; DOI:10.1007/s11033-015-3874-x · 1.96 Impact Factor

Preview

Download
1 Download
Available from